Ring-a dehydrogenation of steroids with nocardia microorganisms



United States This invention is concerned generally with the productron of valuable steroid compounds by fermentation. More particularly, it relates to a novel microbiological method for introducing a double bond between the C-1 and 3-2 carbon atoms of the steroid nucleus by means of microorganisms of the genus Nocardia. In accordance with a preferred embodiment of the invention, A -3- keto-steroids such as A -pregnene-3,l1,20-t-rione-l7e,21- diol and A -pregnene-3,20-dione-11 9,17a,21-triol are converted to the corresponding A -3-keto-steroids, A -pregnadiene-3,1l,20-trione-l7a,2l-diol and .d -pregnadiene- 3,20-dione-11fi,17a,2l-triol, respectively. The latter com pounds possess cortisone activity but differ from cortisone in being substantially free from undesired side effects such as edema since they do not possess any appreciable sodium or water retention action.

The preparation of A -3-keto-steroids by chemical means has been unsatisfactory due to the fact that the chemical reactions involved give mixtures of several compounds. Separation of the desired intermediates and final products from such mixtures is costly and results in the obtainment of low yields of the desired A -3-ketosteroid compounds.

It was an object of the present invention to discover 'a one-step method for introducing a A double bond into steroid compounds, and to achieve this A unsaturation by a selective method which would result in the formation of the desired A -steroid uncontaminated by unwanted by-products.

It was a further object to discover a method whereby A -3-keto-pregnene' compounds could be converted directly, and in high yield to the corresponding A -3-ketopregnadiene compounds.

' it was a still further object to achieve the production of A -3-keto-steroids of the pregnane series directly, and in high yield, from the corresponding 3-keto-pregnane and 3-hydroxy pregnane compounds.

It is now discovered that the selective A dehydrogenation of steroid compounds'can be accomplished by a novel microbiological dehydrogenation procedure which comprises contacting a steroid compound, in particular a C-S unsaturated 3-oxygenated steroid compound, with 50 the dehydrogenating activity of microorganisms of the genus Nocardia, preferably microorganisms belonging to. the species Nocardia blackwellii, Nocardia arteroides, Nocardia minima, Nocardia globerula, Nocardia leishmanii, Nocara'ia formica, Nocardia convoluta, and

' Nocardia corallina. These Nocardia microorganisms can be obtained from known sources such as the American Type Culture Collection, Washington, D.C., or they may be isolated from natural sources, such as soil or disease processes, by known methods.

The dehydrogenation can be effected by contacting the steroid compound with the Nocardia microorganisms themselves or, if preferred, with enzyme systems of the Nocardia microorganisms whereby the hydrogen attached to the C -1 and 0-2 carbon atoms is selectively removed to produce the desired Al-s'teroid substantially uncontaminated by unwanted by-pro ducts. When a A -3-ke'topregnene compound is subjected to the dehydrogenating activity of Nocardia microorganisms the corresponding A -pregnadiene compound is obtained directly and in highyield. v

atm J While this novel microbiological dehydrogenation method is applicable for the A dehydrogenation of 3-keto-steroid compounds generally irrespective of the substituents attached to, or degree of unsaturation in, rings B, C and D, it is ordinarily preferred to utilize, as starting materials in this method, C-5 unsaturated 3-oxygenated-steroid compounds of the pregnane series as for example A -3 keto-pregnene compounds such as A -pregnene-3,2O-dione, A -pregnene-3,20-dione-17a-ol, A -pregnene-3 ,ZO-dione-Zl-ol, A -pregnene-3,20-dione-2l-ol 2l-acylate, -pregnene-3 ,20-dione-21-ol 21 acetate, A -pregnene-3,20-dione-17 a,21diol, M-pregnene-BJO-dione-17,2l-diol 21-acylate, A -pregnene-3,20-dione-17a,2l-diol 2l-acetate, A -pregnene-3,20-dione-17u-ol-2 l-al, A -pregnene-3 ,1 1,20-trione, A -pregnene-3,1 1,20-t1i0I16-17a-0l, A -pregnene-3 ,1 1 ,ZO-t-rione-Z 1-01, A -pregnene-3,11,20-trione-21-ol 21-acylate, A -pregnene-3 ,1 1,20-trione-21-ol 21-acetate, A -pregnene-3,11,2O-trione-l7a,21-diol, A -pregnene-3,11,20-trione-17,21-diol 2 l -acylate, A -pregnene,3,1l,2O-trione-17a,21-dio1 21-acetate, A -pregnene-3,1 1,20-trione-17a-ol-21-al,

-pregnene-3,2O-dione-116,21-01, A -pregnene -3,20-dione-1lfi-diol, M-pregnene-B ,ZO-dione-l 1,21-diol 21-acylate, A -pregnene-3,20- dione-1113,21-diol Zl-acetate, A -pregnene-3,20-dione-1 1 B-ol-Z l-al, A -pregnene-3,2O-dione-1 1 [3,17a-di01, A -pregnene-3,20-dione11B,17a,21-triol, A -pregnene-3,2O-dione-1 1,17,21-triol 21-acylate, A -pregnene-3,20-dione-1 1 [9,1704-(1101-21-31, a A -pregnene-3,20-dione-1 1B,17a,21-triol-2 l-acetate,

9-halo-derivatives of these A -3-keto-pregnene compounds I such as 9-halo-A -pregnene-3 ,20-dione-1 7,21 -diol, 9-fluoro-A -pregnene-3,2O-dione-17u,2 l-diol,

9-halo-A -pregnene-3,20-dione-17,2l-diol 21-acylate, l

, 9-halo-A -pregnene-3,11,20-trione-17,2l-diol 2l-acylate, I 9-fiuoro A pregnene-3,11,20-trione-17a,21-diol 21-acetate, 9-halo-A -pregnene-3,20-dione-1 1,17,2l-triol, 9-fiuor0-A -pregnene-3,20-dione-1L8,17a,21-triol, 9-halo-A -pregnene-3,-2O-dione-11,17,21-triol 21-acylate, 9 fluoro-M-pregnene-S,ZO-dione-l1fl,17a,21-triol 21-acetate,

and the like.

droxy-pregnene compounds such as A -pregnene-20-one-3-ol, A -pregnene-20-one-3,l7a-diol, A -pregnene-20-one-3,2l-diol,

A -pregnene-20-one-3,2l-diol 21-acylate,

'n -pregnene 2O-one-3,17,2l-triol 21-acylate,

A -pregnene-21-one-17 a,2l-triol 21-acetate, A -pregnene-11,2O-dione-3-ol, A -pregnene l 1,20-dione-3,17a-diol, A -pregnene-11,20-dione-3 ,21-diol, A -pregnene-11,20-dione-3,21-diol 2l-acylate,

- -A -pregnene-11,20-dione-3,2-diol ZI-acetate,

Instead of A -3-keto-pregnene compounds," I may employ as starting materials'other C-5 unsaturated 3-oxygenated-steroid compounds as for example A -3 -hy:

M-pregnene-l 1,20-dione-3, 17 :,2 l -triol, A -pregnene-1 1,20-dione-3,17,21-triol 21-acylate, A -pregnene-1 1,20-dione-3,17a,21-tri0l 2 l-acetate, A -pregnene-20-one-3J lfi-diol, A -pregnene20-one3,1 15,21-triol, A -pregnene-20-one-3,1 1,21-triol 21-acylate, A -pregnene-20-one-3 ,1 15,2 l-triol 2 l-acetate, A -pregnene-20-one-3 ,1 1fl,17a-triol, A -pregnene-20-one-3 ,1 1B,17rx,21-t6t10l, A -pregnene-20-one-3,11,17,21-tetrol 21-acylate, M-pregnene-ZO-one-SJ 1fi,17a,21-tetrol 21-acetate, 9-halo-derivatives of these A5-3-hydroxy-pregnene compounds such as 9-halo-A -pregnene-2O-one-3,17,2l-triol, 9-fluoro-A -pregnene-20-one-3,17a,2l-triol, 9-halo-A -pregnene-20-one-3 ,17,21-triol 2 l -acylate, 9-fluoro-A -pregnene-20-one-3 ,17oz,21tfi01 2 1 -acetate, 9-halo-A -pregnenel 1,20-dione-3,17,21-t-riol, 9-fiuoro-A -pregnene-l 113,20-dione-3 1 701,2 l-triol, 9-hal0-A -pregnene-1 1,20-dione-3, 17,21-triol 21-acylate, 9-fluor0-A -pregnene-1 1fi,20-dione-3,17a,2l-triol 21-acetate, 9-halo-A -pregnene-20-one-3,1 1,17,21-tetrol, 9-fluoro-A -pregnene-20-one-3,1 1 8,l7ot,2l-tetrol, 9-halo-A -pregnene-20-one-3,1 1,17,21-tetrol 21-acylate, 9-fluoro-A -pregnene-20-one-3,l 1,6, 1 711,2 l-tetrol 21-acetate, A -3-(lower alkanoyloxy) pregnene compounds such as A -pregnene-20-one-3-ol 3-(lower alkanoate), A pregnene-20-one3-ol 3-acetate, A -pregnene-20-one-3,17-diol 3-(lower alkanoate), A -pregnene-20-one-3,17a-diol 3-acetate, A -pregnene-20-one-3,21-diol 3-(lower alkauoate), A -pregnene-20-one-3,2l-diol 3-aoetate, A -pregnene-2O-one-3,2l-diol 3,21-bis (lower alkanoate) A -pregnene-20-one-3,2l-diol 3,21-diacetate, A -pregnene-20-one-3,17,21-triol 3-(lower alkanoate), A -pregnene-20-one-3 1 7a,2 l-triol 3-acetate, A -pregnene-20-one-3,17,21-trio1 3,21-bis (lower alkanoate), A -pregnene-20-one-3,17a,21-triol 3,21-diacetate, A -pregnene-11,20-dione-3-ol 3-(lower alkanoate) A -pregnene-1l,20-dione13-ol 3-acetate, A -pregnene-l1,20-dione-3,17-diol 3-(lo'wer alkanoate), A -pregnene-11,20-dione-3,17a-diol l t-acetate, A pregnene-l1,20-dione-3,2l-dio-l 3-(lower alkanoate), A -pregnene-11,2O-dione-3,21-diol 3-acetate, A -pregnene-11,20-dione-3 ,21-diol 3 ,21-bis (lower alkanoate), I M-pregnene-ll,20-dione-3,2l-diol 3,2l-diacetate, M-pregnened1,20-dione-3,17,21-triol 3-(lo'wer alkanoate), A -pregnene-1 1,20-dione-3,17a,21-triol 3-acetate, A -pregnene-11,20-dione-3,17,21-triol 3,21-bis(lo-wer alkanoate), A -pregnene-11,20-dione-3 ,17ot,21-triol 3 ,21-diacetate, M-pregnene-ZO-one-BJl-diol 3-(lower alkanoate), A -pregnene-2O-one-3,1lB-diol 3-acetate, A -20-one-3,11,17-tri0l 3-(lower alkanoate), A -20-.one-3,115,,17a-triol 3-acetate, A -pregnene-20-one-3,11,2.1-triol 3-(lower alkanoate), A -pregnene-20-one-3,l1,8,21-triol S-acetate, A -pregnene-20-one-3,11,21-triol 3,2'1-bis(lower alkanoate), A -pregnene-20-one-3,115,21-triol 3,21-diacetate, A -pregnene-2O-one-3,llqt,17,21-tetrol 3-(lower alkanoate), A -pregnene-20-one-3,l1/i,l7m,21-tetrol 3-acetate, A -pregnene-20-one-3,l 1,17,21-tet1ol 3,2l-bis( lower alkanoate), A -pregnene-20-one-3,11fi, 17a,21-tetrol 3,2l-diacetate,

9-halo derivatives of these A -3-(loWer alkanoyl'oxy)- pregnene compounds such as 9-halo-A -pregnene-20-one-3,17,21-triol 3-(lower 'alkanoate),

. 4, 9-fluoro-A -pregnene-20-one-3,17e,21-triol 3-acetate, 9-halo-A -pregnene-ll,20-dione-3,l7,21-triol 3-(lower alkanoate), I 9-fiuoro-A -pregnene-11,20-dione-3,17u,21-triol 3-acetate, 9-halo-A -pregnene-11,20-dione-3,17,21-triol 3,21-bis- (lower alkanoate), 9-fiuoro-A -pregnene-11,20-dione-3 ,17a,21-triol-3 ,21-

diacetate, 9-halo-A -pregnene-20-one-3.,11,17,21-tetrol 3- (lower alkanoate), 9-tluoro-A -pregnene-2O-one-3 ,1 118, 17a,21-t6tr0l 3-acetate, 9-halo-A -pregnene-20-one-3,11,17,21tetrol 3 ,21-bislower alkanoate), 9-fluoro-A -pregnene-20-0ne-3,11B,17a,21-tetrol 3,21-

diacetate, and the like. These A -3-oXygenated-pregnene compound starting materials are converted by the dehydrogenating activity of these Nocardia microorganisms directly to the corresponding A -3-keto-pregnadiene compounds, which conversion may occur, it is believed, through the intermediate formation of the A -3-ketocompound. In the foregoing starting materials, the preferred ester substituent in the 3 and/or Zia-position is ordinarily the. acetate although other lower hydrocarbon carboxylic acid esters such as propionate, butyrate, tertiary butyl acetate, benzoate, and the like may be employed instead of the acetate if desired. Instead of these C-S unsaturated 3-oXygenated-pregnene com-pounds, I can also utilize as starting materials 3-keto-pregnane compounds or 3-hydroXy-pregnane or S-hydroxy allopregnane compounds which are selectively dehydrogenated by the Nocardia microorganisms to produce A and A unsaturation (and in the case of the 3-hydroXy-pregnane compounds, oxidizing the 3-hydroxy radical to a 3-keto substituent), thereby forming the corresponding A -3-keto-pregnadiene compound. The 3-ketopregnanes and 3-hydroxy pregnanes which I ordinarily employ as starting materials for this microbiological dehydrogenated method using Nocardia microorganisms include pregnane-3-20-dione, pregnane-3,20-dione-17ot-ol, pregnane-3,20-dione-2l-ol, pregnane-3,20 dione 170z,21.di01, pregnane 3,11,20 trione, pregnane-3,11, 20-trione 1711-01, pregnane-3 .11,20-trione- 21-01, pregnane-3,ll ,2G-trione-17a,21-diol, pregnane-3,20.

dione-l 1 5-01, pregnane-3,20-dione-l15,17a-diol, pregnane- 3,20-dione-1 15,21 diol, pregnane-3,20-dione-11p,17a,21- triol, pregnane-20-one-3-o-l, pregnane-20-one-3,l'la-dio'l, pregnane 20 one 3-21-diol, pregnane-20-one-3,17a,2ltriol 3,17; diol, pregnane-l1,20-dione-3,21-diol, pregnanc- 11-20-dione-3,17ot,2l-triol, pregnane 2O one-3,11fi-diol,

ptegnane-20,-one-3,11 8,17o-triol, pregnane-ZO one-3, 115,,

2l-triol, pregnane 20-one-3, llfi,17a,21-tetrol and, for those starting materials enumerated herein-above, which contain hydroxy radicals in the 3- and/ or 21-positions, 3- and/or 21. esters thereof with lower hydrocarbon carboxylic acids such as acetic acid, prop' onic acid, tertiary butyl-acetic acid, benzoic acid, and the like.

The presently-invented microbiological dehydrogenation procedure is conducted by contacting the 3.-oxygenatedsteroid compound with the dehydrogenating activity of Nocardia microorganisms. This can be effected by adding the steroid compound as asolid, or as a solution in a solvent as for example a dialkyl ketone such as acetone, a dialkyl-formamide such. as dimethyl-formamide, and the like, under sterile conditions to a culture of the microorganism in a nutrient medium and agitating the resulting, mixture thereby bringing about growth of, the microorganism and dehydrogenation of the steroid compound. The steroid can be added atthe time the nutrient medium is inoculated with the culture of Nocardi'a microorganisms or, alternatively, may be added to an established culture. Instead of adding the steroid compound to the established culture in the nutrient medium, the cell growth from such established culture may be filtered from the broth, washed pregnane-l 1,20-dione-3-ol, pregnene-ll,ZO-dione-v solution containing the '3-oxygenated-steroid compound,

and the resulting mixture agitated thereby effecting dehydrogenation of the steroid compound to form the corresponding A -3-oxygenated-ster0id. The latter is more readily recovered from this medium than from the mixture obtained when the steroid is incubated with the microorganism in the original nutrient medium; also higher yields of A -steroids are obtained with this medium when 3-hydroxy-pregnanes are used as starting materials. Alternatively, the 3-oxygenated-steroid compound may be contacted with dehydrogenating enzyme preparations from the growth of Nocardia microorganisms, thereby forming the corresponding A -3-oxygenated-steroid compound.

The nutrient mediums used in carrying out this microbiological dehydrogenation are those ordinarily utilized in the propagation of Nocardia microorganisms. The usual nutrient include a nitrogen and carbon source, inorganic salts and growth factors when required. The carbon can be provided by compounds such as acetates, lactates, which provides increased growth and higher yields in the case of certain species such as Nocardia blackwellii, and the like. The nitrogen can be provided by an ammonium salt, amino acids, or proteins such as soybeans, oats, yeast, yeast extracts, tryptic digest of casein, meat extract, blood meal, protein meat and bone scrap, salmon meal, fish meals, fish solubles, distillers solubles, and the like. If desired, the Nocardia microorganisms can be propagated using proteins (or amino acids) without any carbohydrate being present in the medium, in which case the proteins or amino acids serve as the source of both the carbon and nitrogen required by the microorganisms.

vWhile, as noted hereinabove, the dehydrogenation of the 3-oxygenated-steroid compound may be carried out using dehydrogenating enzyme preparations from the growth of Nocardia microorganisms, or by contacting the steroid compound with a suspension of an established culture in distilled water, it is ordinarily preferred to add the 3-oxygenated-steroid compound to a nutrient medium containing a 24-hour growth of Nocardia microorganisms. The proportion of steroid compound which may be added to the medium varies depending upon the particular substrate being dehydrogenated, but it is ordinarily preferred to employ a concentration of about 0.005% to 0.2% of 3-ox'ygenated-steroid compound, although higher or lower concentrations may be employed, if desired. The culture containing the added 3-oxygenated-steroid compound is then incubated, preferably with agitation and aeration for an additional period which ordinarily varies between about .10 hours and 50 hours although shorter or longer fermentation times may be advantageous for the dehydro genation of particular 3-keto-steroid substrates. In view of the fact that prolonged fermentations may result in destruction of a portion of the A -3-keto-steroid product, it is ordinarily preferred to employ a fermentation time of about 10 hours to 24 hours which, depending upon the steroid substrate, has been found to result in maximal yields of the A -dehydrogenated steroid product.

After completion of the dehydrogenation process, the product is conveniently recovered from the fermented broth by extraction with a water-immiscible solvent as for example a chlorinated hydrocarbon such as chloroform, a ketone such as methyl isobutyl ketone, an alkyl alkanoate such as ethyl acetate, and the like. The extract of A -dehydrogenated steroid product and any unreacted starting material which may be present is conveniently purified by chromatography using silicagel, activated alumina, and the like or, if desired by means of descending paper chromatograms. After separation of the dehydrogenated product from unreacted starting material, the product can be purified further, if desired by recrystallization from a solvent such as ethyl acetate, ethyl acetatepetroleum ether, and the like, I

In accordance with this microbiological dehydrogenation method, and utilizing the C- unsaturated 3-oxygenated-steroid compounds, S-keto-pregnane compounds and 3-hydroxy-pregnane compounds enumerated hereinabove, there are obtained A -3-keto-pregnadiene compounds such as A -f -pregnadiene-lt,20-dione, A pregnadiene-Zi, 20-dione-l7a-ol, A -pregnadiene-3,20-dione-2l-ol, A

pregnadiene-3,20-dione-17:,2l-diol, A -pregnadiene-3,20-

dione-l7a-ol-2l-al, A -pregnadiene-3,1 1,20-trione, A pregnadiene-3,11,20-trione-17a-ol, A -pregnadiene-3,11, 20-trione-21-ol, A -pregnadiene-3 ,1 1,20-trionel7u,2 ldiol, A pregnadiene 3,1 1,20-trione-17aol-2lsal, A pregnadiene 3,20-dione-l 113-01, A -pregnadiene-3,20-dione 1lfl-ol-2l-al, A Y-pregnadiene-3,20-dione-l13,21-diol, A pregnadiene 3,20 dione-l 1,8,17a-di0l, N -pregnadiene 3,20 dione 1lB,17u,2l triol, 9-halo-A -3-ketopregnadiene compounds such as 9-halo-A -pregnadiene- 3,20 dione-l7,21-diol, 9-fiuoro-A -pregnadiene-3,20-dione 1701,21 diol, 9-halo-n -pregnadiene-3,20-dione-17, 11,21-triol, 9-halo-A -pregnadiene-B,11,20-trione-l7,21- diol, 9-fluoro-A -pregnadiene-3,1 1,20-trione-l7a,2l -diol, 9 luoro A -pregnadiene-3,20-dione-1l5,17a,2l-triol or the corresponding M-pregnene compounds, and the like.

The following examples illustrate methods of carrying out the present invention but it is to be understood that these examples are given for purposes of illustration and not of limitation.

EXAMPLE 1 Fifty milliliters of a nutrient medium are prepared having the following composition:

Cerelose; g l Edamin... -g 1 Cornsteep liquor ml 0.25

Distilled water to make 50 ml.

This medium is adjusted to pH 6.5 with KOH, sterilized I and inoculated with about 2.5 to 5 ml. of a culture of Nocardia asteroides (MA 272; ATCC 9970) microorganisms, and the inoculated culture is then incubated at a temperature of 28 C., with agitation, for a 48-hour period. To the resulting culture is added a solution containing 10 mg. of hydrocortisone (A -pregnene-l lfi,l7a,21-

triol-3,20-dione) dissolved in 0.1 ml. of dimethylform-' agarnide. The culture containing the steroid compound is incubated, with agitation, for an additional period of about 24 hours at 28 C.

The fermentation broth is extracted with four 50 m1. portions of ethyl acetate, and the ethyl acetate extracts are combined and evaporated in vacuo to a volume of about 5 ml. The concentrated solution is then used to prepare streak-paper chromatograms which are developed utilizing formamide as the stationary liquid'phase and chloroform asthe mobile liquid phase. Two bands are secured, one of which (corresponding to the more mobile component) shows the ultra-violet adsorption maximum characteristic of hydrocortisone, and the other (the less having an ultra-violet adsorption maximum at 242 mu.

The paper chromatogram is thoroughly dried, and the band corresponding to the 242 mu adsorption maximum is cut off and extracted with methanol. Themethanol extract is evaporated to dryness in vacuo to give ri -pregnadiene-l1,8,17a2l triol 3,20-dione.

The material ex EXAMPLE 2 Fifty milliliters of a nutrient medium are prepared having thev following composition:

Cerelose g 1 Edaminu g 1 Cornsteep liquor ....ml 0.25

Distilled water to make 50 ml.

This medium is. adjusted tov pH 6.5 with KOH', sterilized. and inoculated with about 2.5. to ml. of a culture of Nocardia minl'ma (MA 292; ATCC' 8674). microorganisms, and the inoculated culture is then incubated at a temperature of 28 C., with agitation, for a 48-hour period. To the resulting culture is added a solution containing 10 mg. of hydrocortisone (A -pregnene-l1B,17a,2ltriol-3,20-dione) dissolved in 0.1 ml. of dimethylformamide. The culture containing the steroidv compound is incubated, with agitation, for an additional period of about 24 hours at 28 C.

The fermentation broth is extracted with four 50 ml. portions of ethyl acetate, and the ethyl acetate extracts are combined and evaporated in vacuo to a volume of about 5 ml; The concentrated solution is then used to prepare streak paper chromatograms which are developed utilizing formamide as the stationary liquid phase and chloroform as the mobile liquid phase. Two bands are secured, one of which (corresponding to the more mobile component) shows the ultra-violet adsorption maximum characteristic of hydrocortisone, and the other (the less mobile component) shows an ultra-violet adsorption maximum at about 242 mu. The paper chromatogram is dried, and the band. corresponding to the 242 mu adsorption is cut off. and extracted with methanol. The material extractedv with methanol is. again subjected to streak-paper chromatography, using paper which has been extracted for 48 hours with methanol, and employing the chloroform-formamide system previously employed. The resulting chromatcgram shows only a trace band corresponding to the. hydrocortisone starting material with the major band having an ultra-violet adsorption maximum at 242 mu. The paper chromatogram is thoroughly dried, and

EXAMPLE 3 Fifty milliliters of a nutrient medium. are prepared having the following composition:

Cerelose g l Fdamin g 1 Cornsteep liquor ml 0.25

Distilled water to make 50 ml. 1 V

This medium is adjusted to pH 6.5 with KOH, sterilized and. inoculated with about 2.5 to 5 ml. of a culture of Nocardia asteroides (MA 289; ATCC 10904) microorganisms, and the inoculated culture is then incubated at a temperature of 28 C., with agitation, for a 48-hour period. To the resulting culture is added a solution containing 10 mg. of hydrocortisone (M-pregnene-l1,3,17a,21- tri'ol-3,20'-dione) dissolved in 0.1 ml. of dimethylformamide. The culture containing the steroid compound is incubated, with agitation, for an additional period of about 24 hours at 23 C;

The fermentation broth is extracted with four 50 ml.- portions of ethyl acetate, and the ethyl acetate extracts are combined and evaporated in vacuo to a volume of about S-ml. The concentrated solution. is then used to prepare streal .-paper chromatograms which are developed utilizing formamide as the stationary liquid phase and chl'oror'orrnas the. mobile liquid phase. Two bands are secure-done of which (corresponding to the more mobile component) shows the ultra-violet adsorption maximum characteristic of hydrocortisone, and the other (the less maximum at about 242 mu.

3. mobile component) shows. an ultra-violet adsorption maximum at about 242 mu; The paper chromatogram is dried, and the band corresponding to the 242 mu adsorption is cut off and extracted with methanol. The material extracted with methanol is again subjected to streakpaper chromatography, using paper which has been extracted for 48 hours with methanol, and employing the chloroform-fcrmamide system previously employed. The resulting chromatogram shows only a trace band corresponding to the hydrocortisone starting material with the major band having an ultra-violet adsorption maximum at 242 mu. The paper chromatogram is thoroughly dried, v and the band corresponding to the 242 mu adsorption maximum is cut oil and extracted with methanol. The methanol extract is evaporated to dryness in vacuo to give n -pregnadiene-l1,8,l7a,21-triol-3,20=dione.

EXAMPLE 4 Fifty milliliters of a nutrient medium are prepared having the following composition:

Distilled, water to make. 50 ml.

This medium is adjusted to: pH 6.5 with KOH, sterilized and inoculated with about 2.5 to 5 ml. ofa culture of Nocardict globerula (MA 280; ATCC 93-56) microorganisms, and the inoculated culture is then incubated at aternperature of 28 C., with agitation, for a 48-hour period. To the resulting culture is added a solution containing 10 mg. of hydrocortisone (At-pregnene-llyfifllh, 2l-triol--3,20-dione) dissolved in- 0.1 ml. of dimethylformamide; The culture containing the steroid compound is" incubated, with agitation, for an additional period of about 24 hours at 28 C.

The fermentation broth is extracted with four 50 ml.- portions of ethyl acetate, and the ethyl acetate extracts are combined and evaporated in vacuo to a volume of about 5 ml. prepare streak-paper chromatograms which are developed utilizing formamide as the stationary liquid phase and chloroform as the mobile liquid phase. Two bands are secured, one of which (corresponding to the more mobile component) shows the ultra-violet adsorption maximum characteristic of hydrocortisone, and the other (the less mobile component) shows an ultra-violet adsorption The paper chromatogram is dried, and the band corresponding to the 242 mu ad"- sorption iscut off and. extracted with methanol. The material extracted with methanol is again subjectedto streakpaper chromatography, using paper which has been extracted for 48 hours with methanol, and employing the chloroform-formamide system previously employed. The

resulting chromatogram shows'only a trace band corresponding to the hydrocorti'sone starting materialjwithv the ma or band: having an ultra-violet adsorption maximum at 242 mu. The paper chromatogram; is thoroughly dried, and the band corresponding to the 242 mu adsorption maximum is cut off and extracted with methanol. The methanol extract is evaporated to dryness in vacuo' to give A -pregnadienerl' 1,6,l7u,2l-triol 3,20-dione EXAMPLE 5 Fifty milliliters of a nutrient medium are prepared havmgthe following composition-2 Distilled water to make 50ml.

This medium is adjusted to pH 6.5 with Ken, sterilized and inoculated with about 2.5.. to v5 ml. of. a culture 0t N ocardia leishmanii (MA. 281.; ATCC. 6855), microorgan- The concentrated solution is then used to 9.. temperature of 28 C.,-with agitation, for a 48-hour period. To the resulting culture is added a solution containing mg. of hydrocortisone (A -pregnene-11/3,17a,2ltriol-3,20-dione) dissolved in 0.1 ml. of dimethylformamide. The culture containing the steroid compound is incubated, with agitation, for an additional period of about 24 hours at 28 C. I

The fermentation broth is extracted with four 50 ml.-

portions of ethyl acetate, and the ethyl acetate extracts are.

combined and evaporated in vacuo .to a volume of about 5 ml. The concentrated solution is then used to prepare streak-paper chromatograms which are developed utilizing formamide as the stationary liquid phase and chloroform as the mobile liquid phase. Two bands are secured, one of which (corresponding to the more mobile component) shows the ultra-violet adsorption maximum characteristic of hydrocortisone, and the other (the less mobile component) shows an ultra-violet adsorption maximum at about 242 mu. The paper chromatogram is dried, and the band corresponding to the 242 mu adsorption is cut off and extracted with methanol. The material extracted with methanol is again subjected to streakpaper chromatography, using paper which has been extracted for 48 hours with methanol, and employing the chloroform-forrnamide system previously employed. The

resulting chromatogram shows .only a trace band corre-' sponding to the hydrocortisone starting material with the major band having an ultra-violet adsorption maximum at 242 mu. The paper chromatogram is thoroughly dried, and the band corresponding to the 242 mu adsorption maximum is cut off and extracted withmethanol. The meth-anol extract is evaporated to dryness in vacuo to give A -pregnadiene-1 1,3,17a,2l-trio1-3,20-dione.

EXAMPLE 6 Fifty milliliters of a nutrient medium are prepared having the following composition:

Cerelose g l Edamin g 1 Cornsteep liquor "ml" 0.25

Distilled water to make 50 ml.

compound is incubated, with agitation, for an additional are combined and evaporated in vacuoto a volume of' about 5 ml. The concentrated solution is then used to prepare streak-paper chromatograms which are developed oughly dried,.and the band co'rresponding to the 242 muadsorption maximum is cut off and extracted with methanol. The methanol extract is evaporated to dryness dione.

EXAMPLE 7 Fifty milliliters of a nutrient medium are prepared having the following composition:

Cerelose g l Edamin g 1 Cornsteep liquor ml 0.25.

Distilled water to make 50 ml.

This medium is adjusted to pH 6.5 with KOH, sterilized and inoculated with about 2.5 to 5 ml. of a cultureof Noam-die convoluta (MA 275; ATCC 4275) microorganisms, and the inoculated culture is then incubated at a temperature of 28 C., with agitation, for a 48-hour.

are combined and evaporated in vacuo to 'a volume of- The concentrated solution is then used to about 5 ml. prepare streak-paper chroma'to'gra'ms which'are developed utilizing formamide as the stationary liquid phase and chloroform as the mobile liquid phase. Two bands are secured, one of which (corresponding to the more mobile component) shows the ultra-violet adsorption maximum characteristic of hydrocortisone, and the other (the less mobile component) shows an ultra-violet adsorption maximum at about 242 mu. The paper chromatogram adsorption is cut off and extracted with methanol.

band corresponding to the hydrocortisone starting material with the major band having an ultra-violet adsorp- The paper chromatogram is tion maximum at 242 mu. thoroughly dried,'and, the band corresponding to the 242 mu adsorption maximum is cut off and extracted with methanol. The methanol extract is evaporatedto dryness in vacuo to give n -pregnadiene-llB,17a,2l-i

triol-3,20-dione.

' 7 -"EXAMPLE 8 Fifty milliliters of a nutrient medium are prepared having the following composition:

Cerelose g 1 Edamin g 1 Cornsteep liquor ml 0.25

utilizing formamide as the stationary liquid phase and Y The material extracted with methanol is again subjected to streak-paper chromatography, using paper which has been extracted for 48 hours with methanol, and employing the chloroform-formamide system previously employed.

Distilled water to make 50 ml.

This medium is adjusted to pH 6.5 with KOH, sterilized and inoculated with about 2.5 to 5 ml. of a culture of Nocardia corallina (MA 277; ATCC 4273) microorganisms, and the inoculated culture isthen incubated at a temperature of 28" C., with agitation, for a 48-hour period. To the resulting culture is added a solution containing 10 mg. of hydrocortisone (M-pregnene- 11B,17a,2l-triol-3,20-dione). dissolved in 0.1 ml. of dimethylformamide. The culturecontaining the steroid compound is incubated, with agitation, for an additional period of about 24 hours at 28 C.

The resulting chromatogram shows only atrace band I corresponding to the hydrocortisone starting material with the major band having an ultra-violet adsorption maximum at 242 mu. The paper chromatogram is thor- The fermentation broth is extracted with four 50 ml.- v

portions of ethyl acetate, and the ethyl acetate extracts The culture containing the steroid prepare streak-paper chromatograms which are developed utilizing formamide as the stationary liquid phase and chloroform as the mobile liquid phase. Two bands are secured, one of which (corresponding'to the more mobile component) shows the ultra-violet adsorption maximum characteristic of hydrocortisone, and the other (the less mobile component) shows an ultra-violet adsorption maximum at about 242 mu.v The paper chromatogram is dried, and the band corresponding to the 242 mu. adsorption is cut ofi and extracted with methanol. The material extracted with methanol is again subjected to streak-paper chromatography, using paper which has been extracted for 48 hours with methanol, and employing thev chloroform-formamide. system previously employed. The. resulting chromatogram show only a trace band corresponding to the hydrocortisone starting material with the major band having an ultra-violet adsorption maximum at 242 mu. The paper chromatogram is thoroughly dried, and the band corresponding to the 242 mu adsorption, maximum is cutv off. and extracted with methanol. The methanol extract is exaporated to dryness in vacuo to give A1 -pregnadicne-11p,17u,21.-triol-3,20-dione.

EXAMPLE 9 Six 50 mL-portions of the edarnin medium described in Examples 110 8 hereinabove are adjusted to pH 6.5, sterilized by heating for 15 minutes in an autoclave at about 120 C., and. each' of. the sterilized mediums is inoculated with a culture of Nocardia blackwellii micro organisms (ATCC 6846). The inoculated cultures are incubated at a temperature. of 28 C., with agitation, for a. period of about 48 hours, and to each of the six resulting cultures is added a solution of 10 mg. of a different 3- keto-steroid compound in 0.1 ml. of dimethylforrnamide. The cultures containing the 3-keto-steroid compounds are then incubated, with agitation, for an additional period of about 24 hours.

Each of the fermentation broths thus obtained is individually extracted with four 50 mL-portions of ethyl acetate, and the ethyl acetate extracts corresponding to each broth. and derived from a particular 3-keto-steroid substrate are combined and evaporated todryness in vacuo. Each of the six residual products thus obtained is dissolved. in acetone and streaked on paper chromatograms. each of which is developed using-the solvent. system indicated for the particular substrate starting material in the. table hereinbelow. The upper bands for each chromatogram, corresponding to the A -dehydro derivatives, are cut. off, individually extracted with methanol and the methanolrextracted material is. again subjected to streak-paper chromatography. The upper band is again cutoff, then dried, extracted with methanol, and the methanol extract is evaporated to dryness in vacuo to give, for each substrate used as starting material, the

particular A -3-keto-steroid' compound indicated in the following table:

Solvent system used for streak paper chromatography Cerelose g 1 Edamin g 1 Corn-steep liquor ml 0.25'

Distilled water to make 50 ml.

This, medium is. adjusted to pH 6.5 with KOH, sterilized and inoculated with about 2.5 to 5 ml. of a culture of Nocara'ia asteroides (MA 272', ATCC 9970)v microorganisms, and the inoculated culture is then incubated at. a temperature of 28 C., with agitation, for a 48-hour period. To the resulting culture is added a solution con taining 10 mg. of pregnane-l113,17a,21-trioi-3,20-dione dissolved in 0.1 ml. of. dimethylformamide. The culture containing the steroid compound is incubated, with agitation, for an additional period of about 12 hours at 28 C.

The fermentation broth is extracted with four 50 ml.- portions of ethyl acetate, and the ethyl acetate extracts are combined and evaporated in vacuo to a volume of about 5 ml. The concentrated. solution is then used to prepare streak-paper chromato-grams which are developed utilizing formam-ide as the stationary liquid phase and chloroform. as the mobile liquid phase. The principal band thus secured shows the ultra-violet absorption maximum characteristic of hydrocortisone; the paper chromatogram is dried, and this band is cut olf and extracted with methanol. The material extracted with methanol is again subjected to streak-paper chromatography, using paper which has been extracted for 48 hours with methanol, and employing the. ChlOIOfOlIlt fOlIllfiIl'lld system previously employed. The resulting chromatogram is thoroughly dried, and the band showing the ultra-violet absorption maximum characteristic of hydrocortisone is again cut ofi? and extracted with methanol. Themethanol extract is evaporated to dryness in vacuo to give hydrocortisone (A pregnene-l 1 [3-,170;,21-triol 3,20dione) The foregoing procedure is repeated using the same edamin medium, the pregnane-l1/3,17m,21-triol-3,20-dioneas the substrate, and an incubation period of 12 hours, but employing, in. place of the Nocardia asteroiaes (MA 272) microorganisms, the following:

Nocardia minima--(MA 292; ATCC 8674) Nocardia globerula-(MA 280; ATCC 9356) Nbcardia leishmanii-(MA 281; ATCC 6855) Nocardia formica-(MA 143; NRRL 2470) Nocardia aster0ides(MA 289; ATCC 10904) Extraction of the resulting fermentation broths and paper chromatography of the dried extracts in a formamidechloroform system gives, in each case, a hand on the. chromatogram corresponding to hydrocortisone. Elution of this band in the manner. previously described gives hydrocortisone, identified. by its ultra-violet absorption spectrum as the principal steroid product. obtained.

Expt. No. Substrate Stationary Mobile Phase A -3-Keto-Sterio'd' Phase Obtained 1 A pregnene -11fl, Formamldea" Ohl0roform A -Pregnadiene- 17oz, 21-trl0I-3,.20- 115, 17a, 2l-trioldione, I 3, 20-di0ne.

2 lregnane-Ilfi, 17a, do do Do.

2I-trio1-3, 20-dione.

3 Pregame-17a, 21-- .-do I 1:]; chloroform A -Pregnadlenedid-3,11, 20-trione- Toluene. 17a, 21-dio1-3, II, 21-acetate. 20-trione.

4 Altopregnanedh, do -do-. Do. 21-di0lV-3, II, 20- manual-acetate.

5 Preguane-Uct, 21 .-...do Benzene A Pregnadienediol-3, 20-dione. 17a, 21-diol-3, 20-

6 -r Allopregnane-Na, .-...do --do Do.

' 21-dio1, 3,.11-dione- 2L-acetate.

aoraese 13 EXAMPLE 11 A nutrient medium is prepared having the following composition:

Cerelose g 68 Edamin g 68 Cornsteep liquor ml 17 Distilled water to make 3400 ml.

This medium is adjusted to pH 6.5 with KOH, sterilized and inoculated with about 200 ml. of a vegetative growth of a culture of Nocardia blackwellii (MA 273; ATCC 6846) microorganisms, and the inoculated culture is then incubated at a temperature of 28 C., with agitation and aeration, for a 48-hour period. To the resulting culture is added a solution containing 0.64 g. of pregame-3,8,- l7ot,2l-triol-l1,20-dione 2l-acetate dissolved in dimethylformamide. he culture containing the steroid compound is incubated, with agitation and aeration, for an additional period of about 24 hours at 28 C.

The fermentation broth is extracted with ethyl acetate and the extract separated and evaporated to dryness. The residual dried product is partitioned between petroleum ether and 70% aqueous methanol, the petroleum ether layer being discarded. The aqueous methanol containing the product is evaporated under reduced pressure to remove the methanol. The resulting water layer is extracted with ethyl acetate several times and the ethyl acetate layer evaporated to dryness to yield partially purified A pregnadiene-l7a,2l-diol-3,l1,20-trione. This material is dissolved in pyridine and reacted in an excess of acetic anhydride to'form A -pregnadiene-17a,2l-diol-3,l1,20-trione 2l-acetate, which is recovered from the acetylation mixture by adding water and recovering the precipitated material by filtration. The A -pregnadiene-17a,2l-diol- 3,11,20-trione 2l-acetate is dissolved in ether and chromatogramed on activated alumina. The alumina column is developed using, as developing solvents, ether and a mixture of ether/chloroform. The desired product is eluted from the column with chloroform, and the chloroform eluate is evaporated to give a high yield of substantially pure, crystalline A -pregnadiene-17a,21-diol-3,ll, 20-trione 2l-acetate.

EXAMPLE 12 Two 50 mL-portions of the edamin medium described 14 with KOH, sterilized by heating for 15 minutes in an autoclave at 120 C., and each of the sterilized mediums is inoculated with a culture of Nocardia blackwellii (ATCC 6846) microorganisms. The inoculated cultures are incubated at a temperature of 28 C., with agitation, for a period of about 48 hours. At the end of the incubation period, the culture growths are centrifuged, the cells being retained and the supernatant liquid discarded. The

retained cells are washed with distilled water and reconstituted to a volume of 25 ml. using water buttered at pH 7.0. To one of the cell suspensions is added a solution of 5 mg. of pregnane-3,l7a,2l-triol-l1,20-dione 2l-acetate in 0.1 ml. of dimethylformamide; to the other cell suspension is added a solution of 5 mg. of pregnane-lhlldiol-3,ll,20-trione 2l-acetate in 0.1 ml. or" dimethylformamide. The cell suspensions containing the pregnane compounds are then incubated, with agitation, for a period of approximately 12-15 hours.

Each of the fermentation broths thus obtained is individually extracted with four 50 mL-portions of ethyl acetate. The ethyl acetate extracts corresponding to each of the two broths are combined and evaporated to dryness in vacuo to give, in each case, relatively pure A -pregnadiene-l7a,21-diol-3,l1,20-trione; yield in both instances, as shown by polographic analysis of the products, is in excess of of theory.

Various changes and modifications may be made in carrying out the present invention without departing from the spirit and scope thereof. Insofar as these changes and modifications are within the purview of the annexed claim, they are to be considered as part of my invention.

I claim:

The process which comprises contacting a 3-keto-pregnane compound with the dehydrogenating activity of Nocardia microorganisms, thereby forming the corresponding A -3-keto-pregnene.

References Cited in the tile of this patent UNITED STATES PATENTS Murray July 8, 1952 Fried et al. July 24, 1956 OTHER REFERENCES 

